Analysis of EGFR Signaling in Single Cells using Capillary Electrophoresis

Abstract

The importance of protein kinases in cell signaling and human disease is well established. While much is understood about how these enzymes regulate the cellular environment, the direct measurement of kinase activity in single intact cells remains challenging. In the present work, a novel assay for measuring Epidermal Growth Factor Receptor (EGFR) activity in single cells has been developed. EGFR is a receptor tyrosine kinase known to play a role in numerous cancers and is implicated in the inflammatory response to air pollution. The analysis of EGFR activity in clinical samples would provide valuable information regarding mechanisms of disease and potential interventions, however these samples tend to be small, heterogeneous mixtures of primary cells that are difficult to analyze using existing technologies that require large cell populations (e.g. Western blotting, flow cytometry) or expression of a genetically encoded sensor (e.g. FRET). We have developed an assay for measuring the phosphorylation dynamics within single intact cells using fluorescent substrate peptides in conjunction with capillary electrophoresis (CE). A peptide reporter is loaded into the cell of interest, phosphorylated by the intact cellular machinery, and then the cell is lysed and its contents analyzed by CE using high sensitivity laser-induced fluorescence detection. The ability to select individual cells coupled with sensitivity to 10−21 mol allows effective analysis of the clinical samples we are interested in studying. This technology has been validated using purified EGFR kinase domain and cell lysates, where the reporter is resistant to degradation for at least 1 hour. Additionally, phosphorylation (16.9% in 90 seconds) has been observed in an intact A431 cell treated with the phosphatase inhibitor sodium pervanadate. Single cell studies are ongoing and will explore pharmacologic modulation of EGFR as well as protein tyrosine phosphatases.